Resistance of high-grade tumors to DNA damage-inducing chemotherapeutic agents (C/Ts) is a major cause of cancer mortality. Hepatocellular carcinoma (HCC) is well known for its innate resistance to C/Ts, but the underlying mechanisms remain unclear. Recent studies are beginning to home in on the notion that the progression and drug resistance of late-stage breast and ovarian cancers may depend on the homologous recombination (HR) repair pathway. My group studies a self-renewal mechanism controlled by a stem and cancer cell-enriched protein called nucleostemin (NS). We recently discovered a key activity of NS in promoting HR repair and protecting cancer cells from replication-induced DNA damage. We also found that NS is highly expressed in Hep3B (human HCC) cells and late-stage diethylnitrosamine (DEN)-induced mouse HCC. The objective of this R03 application is to obtain data to support the clinical relevance of NS in human HCC samples and its potential role in conferring HCC drug resistance via HR repair. We hypothesize that NS expression correlates with the malignancy of human HCC and that NS depletion will compromise the HR- based double-strand break (DSB) repair and chemoresistant property of HCC cells. This hypothesis is formulated based on the following reasons: 1) NS expression is significantly up-regulated in Hep3B cells and in DEN-induced HCC at the late carcinoma stage; 2) NS is required to maintain the continuous proliferation of Hep3B cells; 3) NS promotes HR-based repair and prevents genomic damage in breast (MDA-MB-231) and bone (U2OS) tumor cells; and 4) HR deficiency is known to sensitize breast, ovarian, prostate, and pancreatic cancers to C/Ts. To test our hypothesis, three specific aims will be pursued. Aim #1 will characterize the clinical relevance of NS in human HCC. Aim #2 will determine the effect of NS depletion on HR-based DSB repair in HCC cells. Aim #3 will determine the role of NS in mediating HCC drug resistance. As the outcome of this application, we will establish the clinical importance of NS by showing an increased or dysregulated NS expression in human HCC samples, define the relationship between NS expression and patients' clinical outcome, and provide functional evidence to support the role of NS in promoting the HR-based repair and chemoresistance in HCC cells, all of which are important preliminary data for our future R01 submission. If our hypothesis is proven true, it will impact on our knowledge of how HCC cells adapt to the hyperactive mitotic state and avoid fatal genomic instability at late stage, how HCC develop the innate property of chemoresistance, and whether HCC can be re-sensitized to C/Ts by NS ablation. Given the complexity of hepatocarcinogenesis and heterogeneity of HCC, combining C/Ts with a treatment that targets a common adaptive mechanism used by cancer cells offers an appealing strategy to improve the efficacy and tolerability of C/Ts.